Date of Award

Spring 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Investigative Medicine

First Advisor

PILLAI, MANOJ

Abstract

Recurrent mutations in RNA splicing factor (SF) genes, such as SF3B1, U2AF1, SRSF2, and ZRSR2, are key drivers of oncogenesis in various cancers, particularly clonal myeloid disorders like Myelodysplastic syndromes (MDS) and Acute Myeloid Leukemia (AML). Over the past decade, significant insights have been gained into their molecular mechanisms and effects on RNA splicing. These mutations are typically heterozygous and non-synonymous (except ZRSR2, which involves single-allele loss-of-function mutations) and are largely mutually exclusive, suggesting shared downstream mechanisms that prevent co-occurrence. Given their pre-eminent roles in splicing, most studies to date have primarily focused on the effects of SF mutations on alternative splicing (AS). Accordingly, the enhanced clonal fitness conferred by these mutations arises from aberrant splicing of key oncogenes or tumor suppressor genes altering protein function. However, these AS events are largely mutation-specific and not shared across SF mutation types. This, along with the striking mutual exclusivity of SF mutations, underscores the need to explore broader mechanisms beyond mutation-specific isoform changes in select downstream target genes.Although RNA transcription and splicing are closely coordinated processes, disruption of co-transcriptional splicing as a basis for disease remains unexplored. Transcription studies require studying acute changes in inducible model systems. Furthermore, SF mutations are toxic to cells in vitro, presenting challenges to isolating and growing cells while the mutant protein is actively being expressed. To address this, I developed a novel inducible isogenic cell line system combining AAV-intron trap and CRISPR/Cas9 genome editing. This system enables scalable expansion of cells and inducible mutant allele expression via Cre-recombinase. Generating isogenic models for mutations that confer a growth disadvantage has been challenging. By integrating AAV-intron trap, CRISPR/Cas9, and inducible Cre-recombinase technologies, I achieved >90% efficiency in introducing the oncogenic K700E mutation in SF3B1 and S34F mutation in U2AF1. The AAV-intron trap restricted editing to one allele, while CRISPR/Cas9-directed homologous recombination targeted the desired locus. Inducible Cre-recombinase facilitated cell expansion prior to mutant allele expression, overcoming the toxicity of SF3B1K700E. This optimized approach is adaptable for other oncogenes with similar challenges. Using this inducible model, I discovered that SF3B1 and U2AF1 mutations impair RNA Polymerase II (Pol II) transcription elongation along gene bodies and reduce its promoter density, leading to an increase in R-loop structures, leading to transcription-replication conflicts, replicative stress, and activation of the DNA damage response (DDR). These elongation defects were linked to disrupted pre-spliceosome assembly due to impaired protein-protein interactions of mutant SF3B1, leading to defective early spliceosome complex transitions. This disruption represents a novel disease paradigm for altered transcription dynamics, where co-transcriptional splicing defects directly impact transcriptional elongation. Altered Pol II promoter density was associated with a closed promoter configuration and reduced H3K4me3 marks, attributed to premature Pol II release. An unbiased screen identified epigenetic factors in the Sin3/HDAC/H3K4me pathway as regulators of these defects. Modulating this pathway normalized transcriptional disruptions and their downstream effects, presenting a potential therapeutic target for SF-mutant diseases. One common finding across MDS-associated SF mutations that may explain their mutual exclusivity is the increase in R-loop-associated DDR. However, the downstream consequences of transcription defects, replicative stress, and DDR on fundamental RNA metabolism processes, including alternative splicing (AS), remain poorly characterized. To address this, I investigated the relationship between DDR and altered splicing functions across SF mutants. Analysis of RNA-seq data from 395 SF-mutant patients and 64 healthy donors identified a shared retained intron (RI) signature unique to SF mutations, distinct from healthy controls. These bidirectional RI alterations were highly concordant across mutants, indicating a common trans-acting mechanism rather than mutation-specific cis-effects. The RI patterns correlated with an imbalance in SRSF1 and HNRNP activities. Biochemical analyses revealed that SRSF1 activity loss was due to hypophosphorylation of its C-terminal RS domain. Mechanistic studies in SF-mutant cell lines and patient-derived progenitors implicated disruption of the AKT-SRPK-SRSF1 axis, driven by a loss of AMPK/AKT balance, as the underlying cause of these changes. Notably, activation of the DDR by chemotherapy agents induced similar disruptions in AMPK/AKT balance and SRSF1 hypophosphorylation, linking DDR-induced metabolic stress in clonal states to global signaling pathways regulating pre-mRNA processing and AS. In summary, this study reveals how SF mutations disrupt co-transcriptional splicing and transcription elongation, uncovering a shared retained intron (RI) signature across SF mutations driven by DDR-induced metabolic stress. It also highlights the critical role of DDR in mediating alternative splicing dysfunction through the disruption of key signaling pathways, including the AKT-SRPK1 axis. These insights provide a strong rationale for targeting the Sin3/HDAC complex and related pathways as potential therapeutic strategies to mitigate transcriptional and splicing defects in SF-mutant diseases.

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